Negative-strand RNA viruses (NSVs) include measles, influenza A, Ebola and many other causative agents of serious human disease. In order to gain a better understanding of the replication cycle of this class of viruses, we are studying the molecular structure of vesicular stomatitis virus (VSV), a prototypic non-segmented NSV (NNSV) in the rhabdovirus family. VSV and the other NNSV encode a functionally conserved set of proteins (N, P, and L) that serve to duplicate their genomes and produce functional viral mRNAs. The processes of transcription and replication require two viral components, the RNA-dependent RNA polymerase (RdRp, a complex between L and P) and the genomic template. For NSVs, the active template for both of these processes is the nucleocapsid (N+RNA), not the naked RNA alone. Our previous structural studies showed how the RNA is encapsidated by the nucleocapsid and how the RNA adopts a unique structure once within the capsid. The RdRp must gain access to the bases of the sequestered RNA in order to initiate polynucleotide synthesis. Since the nucleocapsid is the template for viral RNA synthesis and given the intimate association between N and the RNA, questions arise as to what role the N protein may play in transcription and replication. The new studies proposed here are focused on: Aim 1, the structural requirement of the functional template in initiation of viral transcriptin and replication. We have developed methods to solve the structure of specific RNA sequences encapsidated within nucleocapsid-like particles (NLPs). In this aim, we plan to solve a novel series of structures that address the question about how the N protein helps the RdRp to recognize viral specific RNA sequences (such as the 3' genomic termini and transcriptional initiation sequences) within the nucleocapsid. These studies will be complemented with a look at structural changes in the N protein that affect transcription and replication. This will be accomplished by studying a series of mutant N proteins with phenotypes that affect these enzymatic processes. These mutants suggest that the N protein itself plays a role in regulation of transcription and replication. In Aim 2, we will examine polynucleotide synthesis from the role of the L and P proteins. We have engineered a series of L protein fragments that are soluble. The N- and C-terminal regions have already been crystallized with the N-terminal domain diffracting to >2.8 resolution. We will determine structures for several domains of L. These will be integrated with EM studies of the L, P and N proteins aimed at reconstructing the larger tripartite replicase complex and nucleocapsids. Collectively, the studies proposed here will address both replication and transcription of NSV from two perspectives, the template as well as the machinery involved in these essential enzymatic processes. The outcome of our studies may yield information that will promote future drug design strategies against this group of pathogens.